Methods of producing four carbon molecules

ABSTRACT

Disclosed are methods for producing butadiene from one or more of several diverse feedstocks including bioderived feedstocks, renewable feedstocks, petrochemical feedstocks and natural gas.

RELATED APPLICATION

This patent application is a continuation of U.S. application Ser. No.13/524,973, filed Jun. 15, 2012 which claims the benefit of priorityfrom U.S. provisional application No. 61/498,408 filed Jun. 17, 2011.

FIELD OF THE INVENTION

This application is related to a method for producing butadiene from oneor more of several diverse feedstocks including bioderived feedstock,renewable feedstock, petrochemical feedstock and/or natural gas.

BACKGROUND OF THE INVENTION

1,3-Butadiene (hereinafter butadiene) is an important monomer forsynthetic rubbers including styrene-butadiene rubber (SBR), plasticsincluding polybutadiene (PB), acrylonitrile butadiene styrene (ABS),acrylonitrile butadiene (NBR), and as a raw material for adiponitrilefor Nylon-6,6 other chemicals. Butadiene is typically produced as aby-product in the steam cracking process and isolated from the crackerstreams via extraction. On-purpose butadiene has been prepared amongother methods by dehydrogenation of n-butane, dehydrogenation ofn-butane, dehydration of n-butanol or butanediols. Industrially,butadiene has been synthesized using petrochemical-based feedstocks. Thecurrent commercial practices for producing on-purpose butadiene haveseveral drawbacks including high cost of production and low yieldprocesses. Currently, methods for the production of on-purpose butadienerely on petro-chemical feedstocks and on energy intensive catalyticsteps. In this regard, biotechnology offers an alternative approach inthe form of biocatalysis. Biocatalysis is the use of natural catalysts,such as enzymes, to perform chemical transformations on organiccompounds. Both enzymes that have been wholly or partially purified, andenzymes which are present in whole cells are useful catalysts inbiocatalysis.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing intermediates, in particularbutadiene, wherein the methods are biocatalysis based. Both bioderivedfeedstocks and petrochemical feedstocks are viable starting materialsfor the biocatalysis processes.

SUMMARY OF THE INVENTION

The inventors have determined that it is possible to generate enzymeswhich are able to catalyse the conversion of butenols to butadiene.Prior to the inventors' surprising discovery, it was not known thatenzymes capable of introducing double bonds between carbon atoms inhydroxylated unsaturated four carbon molecules existed or could begenerated.

The inventors' discovery is particularly surprising because the reactioncatalysed by the enzymes of the invention is completely contrary to thetypical reaction direction observed in nature. That is, the reaction isin the reverse direction to that which is observed in nature. In nature,double bonds between carbon atoms in a molecule, for example, inunsaturated fatty acids, tend be become saturated, for example, by anenzyme catalysed nucleophilic attack on one of the carbon atoms which isin the double bond. This is, in part, driven by the prevalent conditionsof the intracellular milieu.

Thus, the invention provides enzymes which convert butenols intobutadiene. This conversion can be performed by a single enzyme of theinvention, or may be performed by two or more enzymes of the invention,acting sequentially (that is to say that, for example, a first enzymeacts on a four carbon molecule to produce a first butenol, and thatfirst butenol is then acted upon by a second enzyme of the invention toproduce butadiene). The invention also provides methods of producingbutadiene from a unsaturated hydroxylated four carbon molecule,comprising at least one biocatalytic step. The reactions performed bythe enzymes of the invention include dehydration (i.e. the removal ofH₂O from the molecule)

In some embodiments, the butenol is selected from the group consistingof 1-buten-3-ol, 1-buten-4-ol, 2-buten-1-o1,2-buten-3-ol or2-buten-4-ol.

In some embodiments the butenol can be generated in situ as the enolateof the corresponding unsaturated ketone or aldehyde such as 1-butenal or2-butenal or a 2-keto butene.

In some embodiments, a butenol is produced from four carbon moleculesselected from a butanediol (1,4-butanediol, 1,3-butanediol, or2,3-butanediol) or a butanol (1-butanol, or 2-butanol) or a butene(1-butene or 2-butene) by the action of an enzyme.

In some embodiments, the butenol is produced from a butene such as1-butene or 2-butene.

The reactions performed by the enzymes of the invention will bedehydration (i.e. the removal of H₂O from the molecule), oxidoeductase(i.e. the replacement of a hydrogen with a hydroxyl group), ordehydrogenation (i.e. the removal of hydrogen from the molecule). In thereactions catalysed by the enzymes of the invention, dehydrogenationresults in a desaturation of the carbon backbone of the molecule. Forthe dehydration step the enzyme may be the same enzyme class as theenzyme class used for the dehydration of the butenol to butadiene or maybe of another enzyme class.

In a separate invention, the invention provides an enzyme from theenzyme class 4.2.1.-. Enzymes in this class convert butanediols tobutenol.

In some embodiments the butenol or butandiol can be derived frommicrobial fermentation processes based on biological or non-biologicalfeedstocks. For instance, the butenol or butanediol can be derived fromenzymatic or bioprocesses based on biological feedstocks such asglycerol, Synthesis Gas from biomass, sugars from food stuffs such assucrose or glucose, or sugars from non-food stocks such as cellulosic orhemicellulosic derived sugars. Alternatively, the butenol or butandiolcan be derived from bioprocesses based on non-biological feedstocks suchas Synthesis Gas from coal, natural gas, combustion off-gases, andmunicipal waste or petrochemical derived feedstocks such ashydrocarbons. Further, the butenol or butanediol can be derived fromnon-enzymatic processes based on petrochemical feedstocks.

The reactions performed by the enzymes of the invention will bedehydration (i.e. the removal of H₂O from the molecule).

In a separate invention, the invention provides an enzyme which convertsbutenes to butenols.

The butenol may be produced from four carbon molecules selected from thegroup consisting of a butene such as 1-butene or 2-butene. Further, thebutenol may be selected from the group consisting of 1-buten-3-ol,1-buten-4-ol, 2-buten-1-ol,2-buten-3-ol or 2-buten-4-ol.

The reactions performed by the enzymes of the invention will beoxidoeductase (i.e. the replacement of a hydrogen with a hydroxylgroup).

In another embodiment, the invention provides an enzyme or series ofenzymes which converts butanols to butenols. A butenol is produced fromfour carbon molecules selected from the group consisting of a butanolsuch as 1-butanol or 2-butanol. Additionally, the butenol may beselected from the group consisting of 1-buten-3-ol, 1-buten-4-ol,2-buten-1-ol,2-buten-3-ol or 2-buten-4-ol.

In some embodiments, the butenol is produced directly from the butanolby action of a Cytochrome P450 type enzyme or other desaturase enzymessuch as enzyme class 1.14.99.- or 1.3.1.- or directly from a butanediolby the action of a dehydratase

In some embodiments, the butanol is formed via multiple enzymatic stepsfrom oxidized intermediates such as 1-butanal, butyric acid, or butyricacid CoA prior to reaction with the desaturase enzyme resulting in adesaturaton of the carbon molecule. The unsaturated oxidizedintermediates are thus reduced to a butenol.

In some embodiments the butanols and butanediols can be derived fromenzymatic processes based on biological or non-biological feedstocks.For instance, the butanols and butanediols can be derived from enzymaticprocesses based on biological feedstocks such as glycerol, Synthesis Gasfrom biomass, sugars from food stuffs such as sucrose or glucose, orsugars from non-food stocks such as cellulosic or hemicellulosic derivedsugars. Alternatively, the butanols and butanediols can be derived fromenzymatic processes based on non-biological feedstocks such as SynthesisGas from coal, natural gas, combustion off-gases, and municipal waste orpetrochemical derived feedstocks such as hydrocarbons. Further, thebutanols and butanediols can be derived from non-enzymatic processesbased on petrochemical feedstocks. The reactions performed by theenzymes of the invention will be dehydrogenation of butanols (i.e. theremoval of H₂ from the molecule)or dehydration of butanediols by adehydratase. In the reactions catalysed by the enzymes of the invention,dehydrogenation results in a desaturation of the carbon backbone of themolecule and dehydration results in the removal of a water molecule.

In another embodiment, the invention provides an enzyme or series ofenzymes to produce butadiene from a non-hydroxylated four carbonmolecule selected from the group n-butane, 1-butene, or 2-butene.

The reactions performed by the enzymes of the invention will behydroxylation by a CytP450 enzyme.

The method of the invention can be used with any source of unsaturatedhydroxylated four carbon molecule or its precursor, and therefore issuitable for integration into any known method for synthesisingunsaturated hydroxylated four carbon molecules that can then beconverted into butadiene. For example, the hydroxylated four carbonmolecule may be generated by chemical synthesis or it may be producedbiocatalytically. Methods of synthesising hydroxylated four carbonmolecules are known in the art. Thus the invention provides a method ofsynthesising butadiene from substrates including: syngas, glycerol,CO₂/H₂O, CO₂/H₂, municipal solid waste (MSW), corn, wood pulp,lignocellulose, hemicellulose, macroalgae sugars or sugar, butane,1-butene, 2-butene, n-butanol, iso-butanol, butyricacid,3-butanedio1,2,3-butanediol, 1,4-butanediol, or butenals or 2-ketobutene, comprising at least one enzyme-catalysed step, wherein oneenzyme-catalysed step is the conversion of a butenol to butadiene.

The discovery of a biocatalytic method for the production of butadieneis particularly advantageous because it enables the conversion of fourcarbon molecules to butadiene without the extreme reaction conditionsrequired for chemical catalysis of this reaction, which are highlyenergy intensive.

In one embodiment, the invention involves a method for producingbutadiene by fermentation of a fermentable feedstock. The methodincludes steps of fermenting the fermentable feedstock in the presenceof an organism to produce a fermentation broth comprising aC4-precursor, the precursor including butanol, butanediol, or both. TheC4-precursor is fermented in the presence of the organism to convert atleast a portion of the C4-precursor in the fermentation broth to producebutenol by a pathway comprising: (a) converting butanediol to butenol,or (b) converting butanol to butenol. The butenol is fermented in thepresence of the organism to produce 1,3-butadiene in the fermentationbroth. The 1,3-butadiene is then isolated from the broth.

In another embodiment, the invention involves a method forbiocatalytically producing butadiene from feedstock. The feedstock isconverted in the presence of a biocatalyst into at least a portion of aC4-precursor, the C4-precursor being butanol, butanediol, or both. TheC4-precursor is then reacted with a biocatalyst to convert at least aportion of the C4-precursor in the fermentation broth to produce butenolby a pathway comprising: (a) converting butanediol to butenol, or (b)converting butanol to butenol. The butenol is converted to 1,3-butadienewith a second biocatalyst and then isolated.

In another embodiment, the invention involves a method for producingbutadiene from fermentation of a petrochemical feedstock. The processincludes obtaining butane or butene from the petrochemical feedstock.The butane or butene is then fermented in the presence of an organism toproduce 1,3-butadiene in the fermentation broth, which is then isolated.

In another embodiment, the invention involves a method ofbiocatalytically producing butadiene from a petrochemical feedstock.Butane is obtained from the petrochemical feedstock. The butane iscontacted with a first biocatalyst to produce butene. The butene iscontacted with a second biocatalyst to produce 1,3-butadiene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing pathways for enzymatic butadiene productionaccording to the present invention.

FIG. 2 is a chart showing detailed enzymatic pathways for butadieneproduction from fatty acid, glycerol, and sugar according to the presentinvention.

FIG. 3 is a chart showing detailed enzymatic pathways for butadieneproduction according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention uses one or more enzymes for a specificchemical reaction: the catalysis of the conversion of butenol tobutadiene, the catalysis of the conversion of butanediol to butenol, thecatalysis of the conversion of butene to butenol, the catalyst ofconversion of butanol to butenol, the catalyst of conversion ofunsaturated butyric acid to butadiene, or the catalysis of theconversion of nonhydroxylated four carbon molecules to butadiene.Catalysis by enzymes is highly specific, and thus it is common that asingle enzyme will catalyse only a single reaction, and frequently willcatalyse this reaction with only a low number of substrates. FIG. 1illustrates several catalytic pathways for enzymatic production ofbutadiene according to the present invention.

The catalytic pathway for production of butadiene from fatty acid,glycerol, and sugars is illustrated in FIG. 2. Specifically, fattyacids, glycerols, and sugars may undergo glycolysis and/or fatty acidmetabolism to produce acetyl-CoA. Acetyl-CoA may be converted toAcetoacetyl-CoA through E.C.2.3.1.9. The Acetoacetyl-CoA may then beconverted to (S)-3-Hydroxybutanoyl-CoA by E.C.1.1.1.157. Alternatively,or in addition, 1,2-butanediol may be converted to 3-hydroxybutanalthrough EC 1.1.3.41 and then to (S)-3-Hydroxybutanoyl-CoA through EC6.2.1.-. The conversion of (S)-3-Hydroxybutanoyl-CoA to Crotonyl-CoA mayproceed via EC 4.3.1.17. The Crotonyl-CoA may be converted tovinylacetyl-CoA through EC 5.3.3.3, and conversion of vinylacetyl-CoA to4-Hydroxybutyryl-CoA through EC 4.2.1.120. Further, 4-Hydroxybutyryl-CoAmay be converted to 4-Hydroxybutanal through EC 3.2.1-abtT.

Alternatively, or in addition, fatty acid, glycerol, and/or sugar may beconverted to Succinate and/or 2-Oxoglutarate through the tricarboxilicacid cycle (TCA cycle) as shown in FIG. 2. The succinate may beconverted to via EC 6.2.1.5 and the 2-Oxoglutarate may be converted viaEC 1.2.1.52 to succinyl-CoA. The succinyl-CoA may be converted via EC1.2.1.76 to succinate semialdehyde, which may be converted to4-hydroxybutanoate and optionally to 4-Hydroxybutanal through EC1.1.1.-. Alternatively, or in addition, 4-hydroxybutanoate may beconverted to 4-Hydroxybutyryl-CoA via EC 6.2.1.-, and then to4-Hydroxybutanal through EC 3.2.1-abtT.

As further shown in FIG. 2, 4-Hydroxybutanal may be converted to1,4-butanediol through EC 1.1.1.202. The 1,4-butanediol may then beconverted to 1,3-butadiene through EC 4.2.1.-, adh. The 1,4-butanediolmay then be isolated from the reaction medium. The reactions shown inFIG. 2 may be modified to include pathways shown in FIG. 1. For example1,4-butanediol may be converted to butenol via a dehydratase enzymealternatively or in addition to direct coversion to 1,4-butanediol asshown in FIG. 2. Further, any of the intermediate steps within thereaction chain shown may for the starting point for a commerciallyrelevant production of 1,4-butadiene depending on the availablefeedstock.

FIG. 3 illustrates additional pathways for conversion of variousstarting materials to butadiene. These starting materials includeisobutanol, butane, 2-oxoglutarate, valine, leucine, isoleucine,butanoylphosphate, and/or t-butene. These pathways include theconversion of source materials from a petrochemical and/or natural gasfeedstock such as butane. As with FIG. 2, the pathways shown in FIG. 3may be integrated with other pathways described herein. For example,n-butanol may be converted to a butenol through the action of adesaturase enzyme as illustrated in FIG. 1, or may proceed via thepathway illustrated in FIG. 3 that results in formation of1,4,-butanediol, which is then converted to 1,4-butadiene.

Suitable techniques for identifying, isolating and recombinantlymanipulating enzymes are known in the art.

1.1. Enzyme Catalysed Conversions

The enzymes of the invention catalyse reactions in the conversion ofhydroxylated four carbon molecules to butadiene.

The reactions catalysed by the enzymes of the invention include thedehydration of butenol such as 1-buten-3-ol, 1-buten-4-ol, 2-buten-1-ol,2-buten-3-ol or 2-buten-4-ol.to butadiene.

In an alternate reaction, the reactions catalysed by the enzymes of theinvention include the dehydration of butanediol, such as 1,4-butanediol,1,3-butanediol, and 2,3-butanediol, to butenols such as 1-buten-3-ol,1-buten-4-ol, 2-buten-1-o1,2-buten-3-ol or 2-buten-4-ol. These enzymesmay be the same enzymes capable of converting the butenols to butadieneor different enzymes or enzyme classes.

Thus, by combining these two steps of enzyme reactions it is possible toconvert 1,4-butanediol, 1,3-butanediol, and 2,3-butanediol to butadiene.In this instance, the dehydratase enzyme may act first on the butanediolto produce butenol, which is then acted upon by the same or differentdehydration enzyme to produce butadiene.

In an alternative reaction, a hydrolyase enzyme can be used to introducea hydroxyl group into a non-hydroxylated four carbon molecule. Typicallythe substrate for this reaction will be 1-butene or 2-butene. Here, uponaction of the oxidoreductase enzyme, a hydroxyl group is introducedeither on the terminal carbon or the allylic carbon to produce abutenol. Thus, following reaction with this enzyme, 1-butene isconverted to 1-buten-4-ol or 1-buten-3-ol and 2-butene is converted to2-buten-1-ol (also known as crotonic alcohol). The 1-butene produced bythe desaturation of butane will in turn be acted upon again by theenzyme to produced 1,3-butadiene. 1-butene-3-ol and 1-butene-4-ol may bedehydrated, using an enzyme as detailed above, to produce 1,3-butadiene.

Thus, by combining this oxidoeductase enzyme with the dehydration stepof butenol enzyme it is possible to convert 1-butene and 2-butene tobutadiene. In this instance, the hydrolase enzyme may act first on thebutene to produce butenol, which is then acted upon by the dehydrationenzyme to produce butadiene.

In an alternative reaction, a desaturase enzyme can be used to introducea C═C bond into a saturated four carbon molecule. Typically thesubstrate for this reaction will be butan-1-ol, butan-2-ol, butane or1-butene. Here, upon action of the desaturase enzyme, a C═C bond isintroduced between the terminal carbon and the penultimate carbon(distal to the functional group present on the molecule in the case ofbutan-1-ol, butan-2-ol or 1-butene). Thus, following reaction with thisenzyme, butan-1-ol, butan-2-ol, butane¹ or 1-butene is converted to1-butene-4-ol, 1-butene-3-ol, 1-butene or 1,3-butadiene, respectively.The 1-butene produced by the desaturation of butane will in turn beacted upon again by the enzyme to produced 1,3-butadiene. 1-butene-3-oland 1-butene-4-ol may be dehydrated, using an enzyme as detailed above,to produce 1,3-butadiene.

Thus by combining these two classes of enzymes it is possible to convertbutan-1-ol and butan-2-ol to butadiene. In this instance, thedehydratase enzyme may act first on the butanol to produce 1-butene,which is then acted upon by the desaturase to produce butadiene.

Alternatively, the desaturase may act first to produce 1-buten-3-ol or1-buten-4-ol, which is then reacted to produce butadiene by thedehydratase enzyme.

In an alternate reaction, a desaturase enzyme can be used to introduce adouble bond into a saturated four carbon carboxylic acid or aldehyde.Typically the substrate for this reaction will be butyric acid orbutyraldehyde. Here, upon action of the desaturase enzyme, a C═C bond isintroduced between the terminal carbon and the penultimate carbon(distal to the functional group present on the molecule in the case ofbutyric acid or butyraldehyde). Thus, following reaction with thisenzyme, butyric acid or butyraldahyde is converted to3-butene-carboxylic acid, 2-butene-carboxylic acid, 4-oxo-but-1-ene or4-oxo-but-2-ene, respectively. The resultant unsaturated butyric acid orbutyraldehyde will in turn be acted upon again by an enzyme or series ofenzymes to produce the corresponding butenol. 2-butene-4-ol or1-butene-4-ol may be dehydrated, using an dehydratase as detailed above,to produce 1,3-butadiene. The butyric acid and the butyraldahyde can beproduced enzymatically from 1-butanol by action of an oxidase enzyme.Thus by combining these series of reactions 1-butanol can be convertedto butadiene.

Enzymes suitable for use in the methods of the invention

1.1.1. Dehydratase Enzymes

Dehydratases in EC 4.2.1.- can be used to catalyse a number of steps ofreactions which convert butenols to butadiene and/or butanediols tobutenols. See FIGS. 2-3.

Dehydratses according to the invention comprises enzymes which arecapable of:

a) dehydrating 1-butene-3-ol to produce butadiene;

b) dehydrating 1-butene-4-ol to produce butadiene;

c) dehydrating 2-butene-1-ol to produce butadiene.

d) Dehydrating 2-buten-3-ol to produce butadiene

e) Dehydrating 2-butene-4-ol to produce butadiene

f) dehydrating 1,4-butanediol to produce 1-buten-4-ol;

g) dehydrating 1,3-butanediol to produce 1-buten-3-ol, 1-buten-4-ol, or2-buten-4-ol; or

h) dehydrating 2,3-butanediol to produce 1-buten-3-ol or 2-buten-3-ol

1.1.2. Desaturase Enzymes

Desaturase enzymes of the invention introduce a double bond inton-butanol or iso-butanol at the saturated terminal carbon. Desaturaseshave been demonstrated in the prior art to introduce double bonds atspecific positions in fatty acids. Furthermore, it is possible to modifythe substrate- and regio-specificities of these enzymes. See Wang etal., “Alteration of Product Specificity of Rhodobacter sphaeroidesPhytoene Desaturase by Direct Evolution,” J. Biolog.Chem., Vol. 27, No.44, Issue of November 2, pp. 41161-41164 (2001).

In particular, enzymes in the class EC 1.14.19.- have been found to beuseful in performing the methods of the invention. Other enzymes thatare capable of introducing double bonds into four carbon moleculesinclude members of EC 1.14.99.-, such as 1.14.99.19/30/31/32/33. Enzymesin the class EC 1.3.1.35 are also capable of introducing double bonds.Accordingly, in some embodiments of the invention, the enzyme is inclass EC 1.14.19.-, 1.14.99.-, for example 1.14.99.19, 1.14.99.30,1.14.99.31, 1.14.99.32, 1.14.99.33, or 1.3.1.35.

1.1.3. Cytochrome P450 Enzymes

Aliphatic desaturation can also be catalysed by cytochrome P450 enzymes.Accordingly, in some embodiments of the invention the enzyme is acytochrome P450. The CYP4 isozyme had been reported to catalyse terminaldesaturation of valproic acid to form the 4-ene acid with high activitycompared to CYP2. See Rettie et al., “CYP4 Isozyme Specificity and theRelationship between ω-Hydroxylation and Terminal Desaturation ofValproic Acid,” Biochemistry, 34, 7889-7895 (1995).

1.1.4. Clavaminate Synthase 2

Like P450 enzymes, clavaminate synthase 2 can switch betweenhydroxylation and desaturation, depending on the substrate.2-Oxogluterate-dependent non-heme iron enzymes of the clavaminatesuperfamily are thus also capable of introducing terminal double bondsin alkanes, alkenes, alkenols and alkenoic acids. In particular,clavaminate synthases of the class EC 1.14.11.22 are capable ofconverting hydroxylated four carbon molecules to butadiene. Accordingly,in some embodiments of the invention, the enzyme is in class EC1.14.11.22.

1.1.5. Non-Naturally Occurring Enzymes

In some embodiments, the enzymes used to perform conversions in themethod of the invention are non-naturally occurring. That is to say theDNA encoding them has been mutated from the wild type sequence in orderto improve one or more of the enzyme's properties. Methods formutagenesis of proteins are well known in the art. Random and/orcombinatorial mutagenic approaches may alternatively or additionally beused for the creation of libraries of mutations, including approachessuch as DNA shuffling, STEP and error prone PCR, molecular evolution andmutator strains. A non-limiting list of mutagenic changes includesdeletions, insertions, substitutions, rearrangements, point mutationsand suppressor mutations. The products of the mutagenic methods shouldthen be screened for the desired activity. Thus in some embodiments theenzyme of the invention is derived from an enzyme as described insections. By “derived” is meant that the enzyme contains one or moreamino acid changes compared to the sequence of the wildtype enzyme,wherein the one or more changes includes deletions, insertions,substitutions, rearrangements, point mutations. The skilled person wouldunderstand that the EC classification system discussed in relation tothe enzymes as described is highly specific, and depends on the specificsubstrates catalysed by an enzyme. Accordingly, an enzyme of theinvention derived from one of the enzymes as described may be classifiedin a different EC category to wild type enzyme.

1.2. Biocatalyst Formatting

Whole cells that express one or more of the enzymes of the invention maybe used as the biocatalyst. The whole cells that are used typicallypossess a number of properties: they may be easily genetically modified,are tolerant of the conditions used in the method of the invention, andgrow to cells densities which are industrially useful.

In one alternative, the whole cell is a prokaryote. In anotheralternative it is a eukaryote. Typically single celled microorganismsare used.

The term prokaryotic cell includes gram positive and gram negativebacteria. Examples of gram negative bacteria which may be used with themethods of the invention include: Escherichia coli, Rhodopseudomonaspalustris, sphingomonads, pseudomonads, and other bacteria belonging toSalmonella, Burkholderia, Moraxella, Acaligenes, Psychrobacter,Thermotoga, Acinetobacteria, Rhodobacter, Azoarcus, and Rhodospirillumgenera. Examples of gram positive bacteria which may be used with themethods of the invention include: streptococci, lactobacilli, and otherbacteria belonging to Nocardia, Bacillus, Rhodococcus, Clostridium,Streptomyces, and Arthobacter genera.

Eukaryotic host cells include those from yeast and other fungi. Examplesof eukaryotic host cells which may be used with the methods of theinvention include: Yarrowia lipolytica, Candida genera such as Candidatropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C.maltosa, C. parapsilosis, C. zeylenoides, yeasts belonging to theRhodotorula, Rhizopus, Trichosporon, and Lipomyces genera, and otherfungi belonging to Aspergillus, Exophiala, Mucor, Trichoderma,Cladosporium, Phanerochaete, Cladophialophora, Paecilomyces,Scedosporium, and Ophiostoma genera.

1.3. Modification of Whole Cell Biocatalysts

The biocatalysts used in the methods of the invention may be unmodifiedwhole cells of the species in which the enzyme naturally occurs.Typically, however, it is necessary to modify genetically the host cell.In one alternative, the genetic modification is the introduction of anucleic acid into the genome of the cell. The nucleic acid introducedinto the cell may comprise a nucleic acid sequence from another speciesor organism, for example a DNA sequence that is not present in thewildtype genome of the whole cell. In other instances, the introducedDNA sequence may be a further copy of a DNA sequence in the genome ofthe whole cell. In some alternatives, the genetic modification is thedeletion of DNA sequence from the genome of the whole cell. In anotheralternative, the genetic modification is the modification of the genomeof the cell.

1.4. Use of the Enzymes of the Invention in Whole Cells Engineered toProduce Hydroxylated Four Carbon Molecules

Nucleic acids encoding the enzymes of the invention can be placed intoknown host cells which are capable of producing hydroxylated four carbonmolecules, either as a product or an intermediate in the production ofother compounds. By the extension or diversion of the biosyntheticpathways in these previously known host organisms engineered to producehydroxylated four carbon molecules from renewable feedstocks such ascarbohydrates and fatty acids, as well as from glycerol, syngas orphotosynthesis. These pathways are extended or diverted to furtherconvert the hydroxylated four carbon molecules or precursors thereof tobutadiene.

1.5. Metabolic Engineering of Whole Cells

Metabolic engineering is the process of optimising the parameters in awhole cell in order to increase the ability of a cell to produce acompound. The whole cells used in the method of the present inventionoptionally have been engineered to optimise the output of the butadiene.

1.6. Growing Whole Cell Biocatalysts

In some embodiments of the invention whole cell biocatalysts are usedwhich are growing (i.e. dividing) at the time the whole cells performthe conversions in the method of the invention. In these embodiments thecells are cultured under conditions which optimise the production ofdesired product (i.e. butadiene) or precursor (butonol or buitanediol).As used herein, the term culture is equivalent with fermentor andbioreactor.

1.7. Feedstocks for Process

In some embodiments the butadiene can be derived from enzymaticprocesses based on biological or non-biological feedstocks.

In some embodiments, the butadiene can be derived from enzymaticprocesses based on biological feedstocks such as glycerol, Synthesis Gasfrom biomass, sugars from food stuffs such as sucrose or glucose, orsugars from non-food stocks such as cellulosic or hemicellulosic derivedsugars.

In some embodiments the butadiene can be derived from enzymaticprocesses based on non-biological feedstocks such as Synthesis Gas fromcoal, natural gas, combustion off-gases, and municipal waste orpetrochemical derived feedstocks such as hydrocarbons.

In some embodiments, the butadiene can be derived from non-enzymaticprocesses based on petrochemical feedstocks.

1.8. Compositions of the Invention

The invention also provides compositions comprising an enzyme of theinvention and a four carbon molecule. The invention further providescompositions comprising an enzyme of the invention and 1,3-butadiene.

1. A method for biosynthesizing butadiene comprising: fermenting in an organism in a fermentation broth, a C4-precursor, wherein said C4-precursor comprises butanol, butanediol, or both, or derivatives thereof; converting C4-precursor in the fermentation broth into butenol by: (a) converting butanediol to butenol, or (b) converting butanol to butenol; converting the butenol into 1,3 butadiene; and optionally isolating said 1,3-butadiene.
 2. The method of claim 1, wherein the fermentation broth comprises a biologically derived fermentable feedstock or a nonbiologically derived fermentable feedstock.
 3. The method of claim 2, wherein the biologically derived feedstock comprises glycerol, synthesis gas, synthesis gas from biomass, sugar, sugar from a food stuff, sugar from a non-food stuff, or combinations thereof.
 4. The method of claim 3, wherein the sugar is sucrose, glucose, or combinations thereof.
 5. The method of claim 3, wherein the sugar from a non-food stuff is cellulosic or hemi-cellulosic sugar or combinations thereof.
 6. (canceled)
 7. The method of claim 2, wherein the non-biologically derived feedstock is synthesis gas from coal, natural gas, combustion off-gases, municipal waste, petrochemical, or combinations thereof.
 8. (canceled)
 9. The method of claim 1, wherein the organism is a prokaryote or eukaryote.
 10. The method of claim 1, wherein the C4 precursor is a hydroxylated C4 molecule.
 11. The method of claim 1, wherein converting the butanediol to butenol requires at least one polypeptide having an activity of at least one enzyme.
 12. The method of claim 11, wherein the at least one enzyme is a dehydratase, desaturase or hydrolase enzyme.
 13. The method of claim 11, wherein the at least one enzyme is a polypeptide having an activity of an enzyme EC 4.2.1, EC 1.14.19, EC 1.14.99, EC 1.3.1.35, cytochrome P450, or EC 1.14.11.22. 14-17. (canceled)
 18. The method of claim 1, wherein the butanediol is 1,4-butanediol, 1,3-butanediol, or 2,3-butanediol or derivatives thereof.
 19. The method of claim 1, wherein the butenol is 1-buten-1-ol, 1-buten-2-ol, 1-buten-3-ol, 1-buten-4-ol, 2-buten-1-ol, 2-buten-2-ol, 2-buten-3-ol, or 2-buten-4-ol or derivatives thereof.
 20. (canceled)
 21. The method of claim 1, wherein the organism expresses at least one polypeptide having an enzyme activity capable of converting the C4 precursors to butadiene.
 22. The method of claim 21, wherein the organism is recombinantly engineered to express the polypeptide having the enzyme activity.
 23. The method of claim 21, wherein the polypeptide has the enzyme activity of a dehydratase, a desaturase, or both. 24-35. (canceled)
 36. A method for biosynthesising butadiene from a petrochemical feedstock comprising: obtaining a petrochemical feedstock comprising butane; fermenting butane in the presence of an organism to produce a fermentation broth comprising butene; and fermenting the butene in the presence of an organism to produce 1,3-butadiene in the fermentation broth; and optionally obtaining the 1,3-butadiene.
 37. (canceled)
 38. A microorganism genetically modified to produce 1,3-butadiene.
 39. The microorganism of claim 38 wherein the organism is a prokaryote or eukaryote.
 40. The microorganism of claim 38 recombinantly engineered to express at least one polypeptide having an activity of an enzyme capable of converting C4 precursors to butadiene.
 41. The microorganism of claim 40 wherein the polypeptide having the activity of the enzyme is a dehydratase, desaturase or hydrolase enzyme.
 42. The microorganism of claim 41 which comprises a heterologous nucleic acid sequence encoding a polypeptide having a enzyme activity of an enzyme EC 4.2.1, EC 1.14.19, EC 1.14.99, EC 1.3.1.35, cytochrome P450, or EC 1.14.11.22.
 43. The microorganism of claim 41 which comprises heterologous nucleic acid sequences encoding a polypeptide having an enzyme activity of a dehydratase enzyme classified under EC 4.2.1.- and a desaturase enzyme selected from the group consisting of cytochrome P450 enzymes, clavaminate synthase 2 enzymes and enzymes classified under EC 1.14.19-, EC 1.14.99-, EC 1.3.1-.
 44. A bio-derived, bio-based or fermentation-derived product, wherein said product comprises: i. a composition comprising at least one bio-derived, bio-based or fermentation-derived compound obtained according to claim 1 or any combination thereof, ii. a bio-derived, bio-based or fermentation-derived polymer comprising the bio-derived, bio-based or fermentation-derived composition or compound of i., or any combination thereof, iii. a bio-derived, bio-based or fermentation-derived resin comprising the bio-derived, bio-based or fermentation-derived compound or bio-derived, bio-based or fermentation-derived composition of i. or any combination thereof or the bio-derived, bio-based or fermentation-derived polymer of ii. or any combination thereof, iv. a molded substance obtained by molding the bio-derived, bio-based or fermentation-derived polymer of ii. or the bio-derived, bio-based or fermentation-derived resin of iii., or any combination thereof, v. a bio-derived, bio-based or fermentation-derived formulation comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i., bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., or bio-derived, bio-based or fermentation-derived molded substance of iv, or any combination thereof, or vi. a bio-derived, bio-based or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i., bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., bio-derived, bio-based or fermentation-derived formulation of v., or bio-derived, bio-based or fermentation-derived molded substance of iv., or any combination thereof.
 45. A non-naturally occurring biochemical network comprising at least one substrate of FIG. 1, FIG. 2 or FIG. 3 and at least one exogenous nucleic acid encoding a polypeptide having the activity of at least one enzyme of FIG. 1, FIG. 2 or FIG.
 3. 46. A non-naturally occurring composition, comprising at least one substrate of FIG. 1, FIG. 2 or FIG. 3, wherein said substrate is optionally bio-based, bio-derived or fermentation derived; at least one exogenous nucleic acid encoding a polypeptide having the activity of at least one enzyme of FIG. 1, FIG. 2 or FIG. 3; and at least one bio-based, bio-derived or fermentation derived product of FIG. 1, FIG. 2 or FIG.
 3. 